Leucine-Rich Repeat Kinase 2 (LRRK2) Stimulates IL-1ß-Mediated Inflammatory Signaling through Phosphorylation of RCAN1.

Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase having mixed lineage kinase-like and GTPase domains, controlling neurite outgrowth and neuronal cell death. Evidence suggests that LRRK2 is involved in innate immune response signaling, but the underlying mechanism is yet unknown. A novel protein inhibitor of phosphatase 3B, RCAN1, is known to positively regulate inflammatory signaling through modulation of several intracellular targets of interleukins in immune cells. In the present study, we report that LRRK2 phosphorylates RCAN1 (RCAN1-1S) and is markedly up-regulated during interleukin-1ß (IL-1ß) treatment. During IL-1ß treatment, LRRK2-mediated phosphorylation of RCAN1 promoted the formation of protein complexes, including that between Tollip and RCAN1. LRRK2 decreased binding between Tollip and IRAK1, which was accompanied by increased formation of the IRAK1-TRAF6 complex. TAK1 activity was significantly enhanced by LRRK2. Furthermore, LRRK2 enhanced transcriptional activity of NF-κB and cytokine IL-8 production. These findings suggest that LRRK2 might be important in positively modulating IL-1ß-mediated signaling through selective phosphorylation of RCAN1.

Proteomic analysis reveals upregulation of calreticulin in murine dopaminergic neuronal cells after treatment with 6-hydroxydopamine.

We have utilized integrated technologies including separation of proteins by 2-dimensional (2-D) gel electrophoresis and identification of proteins by matrix assisted laser desorption/ionizing time of flight (MALDI-TOF) mass spectrometry to examine an array of proteins that are regulated following treatment with neurotoxin. In essence, total cellular lysates harvested from MN9D dopaminergic neuronal cells treated with 6-hydroxydopamine (6-OHDA) for various time periods were subjected to 2-D gel separation followed by an analysis of the protein spots separated. Among the several protein spots that appeared to be either up- or down-regulated following 6-OHDA treatment, MALDI-TOF mass spectrometry revealed that an ER chaperone protein, calreticulin, was upregulated in a time-dependent manner. 6-OHDA-mediated up-regulation of this protein spot was reversed to the untreated control level when MN9D cells were co-treated with a pan-caspase inhibitor or an anti-oxidant. Immunoblot analysis using anti-calreticulin antibody confirmed this phenomenon. Since accumulation of altered proteins may be relevant in Parkinson's disease, our data suggest that regulation of chaperone activity in dopaminergic neurons comprises an additional cellular response to death-inducing stimuli.